Background: Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) are among the most complex health problems in the world. Young people are at high risk of HIV and AIDS infections and are, therefore, in need of targeted prevention. School-based HIV/AIDS health education may be an effective way to prevent the spread of AIDS among adolescents. Methods: The study was a school-based intervention conducted in three middle schools and two high schools in Wuhan, China, which included 702 boys and 766 girls, with ages from 11 to 18 years old. The intervention was a one-class education program about HIV/AIDS for participants. HIV/AIDS knowledge, attitude, and high-risk behaviors were investigated using an anonymous self-administered questionnaire before and after the education intervention. Chi-square test was used to compare differences before and after the intervention. Non-conditional logistic regression analysis was used to identify the factors that affect HIV/AIDS knowledge. Results: Misconceptions about basic medical knowledge and non-transmission modes of HIV/AIDS among all the students prevail. Approximately 10% to 40% of students had negative attitudes about HIV/AIDS before the intervention. After the intervention, all of the students had significant improvements in knowledge and attitude about HIV/AIDS (P<.05), indicating that educational intervention increased the students’ knowledge significantly and changed their attitudes positively. Logistic regression analyses indicated that before the intervention the students’ level of knowledge about HIV/AIDS was significantly associated with grade, economic status of the family, and attitudes toward participation in HIV/AIDS health information campaigns. Conclusions: HIV/AIDS education programs were welcomed by secondary students and positively influenced HIV/AIDS-related knowledge and attitudes. A systematic and long-term intervention among secondary school students must be conducted for the prevention of HIV.